Enzyme-based procedures for amplifying polynucleotides are now established tools for diagnostic, environmental and forensic testing. The market for DNA probe diagnostics in clinical laboratories now represents several hundred million dollars each year. The clinical diagnostics-probe business is expected to grow with viral screening and viral load determination representing major areas of active market expansion. Given the commercial value of this technology, great efforts have been invested in research and development of improved amplification procedures (see Genetic Engineering News 17:6 (1997)).
Recently developed techniques for amplifying analyte polynucleotides have provided useful alternatives to methods based on the original Polymerase Chain Reaction (PCR) protocol. According to one technique, DNA amplification reactions are performed on solid-phase substrates made alternatively of glass, plastic, a semiconductor chip or a fiber-optic array. Labeled target DNA is synthesized as a molecular bridge between pairs of oligonucleotide primers immobilized to the solid substrate such that the amplification products remain attached to the solid substrate. U.S. Pat. No. 5,399,491 discloses a different technique wherein a target polynucleotide is amplified autocatalytically under conditions of substantially constant temperature, ionic strength and pH. This method, termed Transcription Mediated Amplification (TMA), allows for the synthesis of multiple RNA copies of target sequence. New methods likely to emerge in the future will continue to expand the range of applications that can be addressed by polynucleotide amplification techniques.
Quantitative amplification assays represent one subset of assays that impose stringent requirements on all aspects of the procedure, including template isolation and standardizing amplification efficiency. Approaches that employ internal standards that participate in amplification reactions are intended to normalize reaction efficiency, but fail to account for variable levels of analyte polynucleotide input into the reaction. Related methods that simultaneously amplify an analyte polynucleotide and control polynucleotides derived from constitutively expressed housekeeping genes also are imperfect because multiple primer sets are required to carry out the amplification reaction.
One example of methods based on the use of internal standards in quantitative PCR amplifications is disclosed in U.S. Pat. No. 5,219,727. According to the method disclosed in this patent, the internal standard is included in the amplification reaction and is designed so that it will amplify at a similar efficiency as the target polynucleotide. Like methods that co-amplify constitutively expressed gene products for use as internal standards, the method disclosed in U.S. Pat. No. 5,219,727 requires detecting and quantifying the amplicon derived from the internal standard in order to quantify the analyte polynucleotide. Thus, several steps still are required to quantitate analyte polynucleotides when an internal standard must be detected and quantitated.
The fact that amplified polynucleotides (“amplicons”) in conventional PCR and TMA procedures are synthesized as molecules free in solution represents another source of inaccuracy for analyte detection. These amplicons can easily be transferred between samples to produce false-positive results in the contaminated reactions. Standard precautions for minimizing false-positive results due to contamination by carried-over DNA templates include ultraviolet irradiation of pipetting devices, the use of disposable glass- and plastic-ware, use of separate laboratories or laboratory areas for conducting amplification reactions, and avoiding the formation of aerosols. One elaborate approach for ensuring that PCR products cannot be re-amplified in subsequent reactions involves a series of steps using specialized reagents to degrade the products from previous PCR amplifications. However, this procedure is somewhat complicated and involves first substituting dUTP for dTTP in the PCR mixture and then pre-treating all subsequent PCR mixtures with a uracil N-glycosylase (UNG) enzyme prior to PCR amplification. Products from previous PCR amplifications are then eliminated by excising uracil residues using UNG, and degrading the resulting abasic polynucleotide (Longo, et al., Gene 93:125 (1990)). Clearly, these methods do not lend themselves to high throughput assays.
Accordingly, there exists a continuing need for techniques that can be used to enhance the precision of polynucleotide amplification procedures. Further, there exists a need for techniques that can be used to diminish the incidence of false-positive results arising from positive carry-over contamination. The present invention addresses both of these needs.